The rapid entry of calcium into cells is mediated by a class of proteins called voltage-gated calcium channels. Calcium channels are a heterogeneous class of molecules that respond to depolarization by opening a calcium-selective pore through the plasma membrane. The entry of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression. In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Miller, R. J., “Multiple calcium channels and neuronal function.” Science (1987) 235:46-52. Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitter. Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and has been implicated in long-term changes in neuronal activity.
In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. Recently, mutations identified in human and mouse calcium channel genes have been found to account for several disorders including, familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. Fletcher, et al. (1996) “Absence epilepsy in tottering mutant mice is associated with calcium channel defects.” Cell 87:607-617; Burgess, et al., “Mutation of the Ca2+ channel β subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (1h) mouse.” Cell (1997) 88:385-392; Ophoff, et al., “Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. ” Cell (1996) 87:543-552; Zhuchenko, O., et al., “Autosomal dominant cerebellar ataxia (SCA6) associated with the small polyglutamine expansions in the α1A-voltage-dependent calcium channel.” Nature Genetics (1997) 15:62-69.
The clinical treatment of some disorders has been aided by the development of therapeutic calcium channel antagonists. Janis, et al. (1991) in Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance. CRC Press, London.
Native calcium channels have been classified by their electrophysiological and pharmacological properties as T, L, N, P and Q types (for reviews see McCleskey, et al., “Functional properties of voltage-dependent calcium channels.” Curr. Topics Membr. (1991) 39:295-326, and Dunlap, et al., “Exocytotic Ca2+ channels in mammalian central neurons.” Trends Neurosci. (1995) 18:89-98.). T-type (or low voltage-activated) channels describe a broad class of molecules that activate at negative potentials and are highly sensitive to changes in resting potential. The L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them. L-type channels are sensitive to dihydropyridine (DHP) agonists and antagonists, N-type channels are blocked by the Conus geographus peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta. A fourth type of high voltage-activated Ca channel (Q-type) has been described, although whether the Q- and P-type channels are distinct molecular entities is controversial (Sather, et al., “Distinctive biophysical and pharmacological properties of class A (B1) calcium channel α1 subunits.” Neuron (1993) 11:291-303; Stea, et al., “Localization and functional properties of a rat brain α1A calcium channel reflect similarities to neuronal Q- and P-type channels.” Proc Natl Acad Sci (USA) (1994) 91:10576-10580; Bourinet, E., et al., Nature Neuroscience (1999) 2:407-415). Several types of calcium conductances do not fall neatly into any of the above categories and there is variability of properties even within a category suggesting that additional calcium channels subtypes remain to be classified.
Biochemical analyses show that neuronal high-threshold calcium channels are heterooligomeric complexes consisting of three distinct subunits (α1, α2δ and β) (reviewed by De Waard, et al., in Ion Channels, (1997) Volume 4, edited by Narahashi, T. Plenum Press, New York). The a, subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel antagonists. The mainly extracellular Alternatively, the α2 subunit is disulphide-linked to the transmembrane δ subunit and both are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the α1 subunit. A fourth subunit, γ, is unique to L-type Ca channels expressed in skeletal muscle T-tubules. The isolation and characterization of γ-subunit-encoding cDNAs is described in U.S. Pat. No. 5,386,025 which is incorporated herein by reference.
Molecular cloning has revealed the cDNA and corresponding amino acid sequences of six different types of α1 subunits (α1A, α1B, α1C, α1D, α1E and α1S) and four types of β subunits (β1, β2, β3 and β4) (reviewed in Stea, A., Soong, T. W. and Snutch, T. P. (1994) “Voltage-gated calcium channels.” in Handbook of Receptors and Channels. Edited by R. A. North, CRC Press). A comparison of the amino acid sequences of these α1 subunits is included in this publication, which is incorporated herein by reference. PCT Patent Publication WO 95/04144, which is incorporated herein by reference, discloses the sequence and expression of as α1E calcium channel subunits.
As described in Stea, A., et al. (1994) (supra), the α1 subunits are generally of the order of 2000 amino acids in length, ranging from 1873 amino acids in α1S derived from rabbit to 2424 amino acids in α1A derived from rabbit. Generally, these subunits contain 4 internal homologous repeats (I-IV) each having six putative alpha helical membrane spanning segments (S1-S6) with one segment (S4) having positively charged residues every 3rd or 4th amino acid. There are a minority of a splice variant exceptions. Between domains II and III there is a cytoplasmic domain which is believed to mediate excitation-contraction coupling in α1S and which ranges from 100-400 amino acid residues among the subtypes. The domains I-IV make up roughly ⅔ of the molecule and the carboxy terminus adjacent to the S6 region of domain IV is believed to be on the intracellular side of the calcium channel. There is a consensus motif (QQ-E-L-GY-WI-E) (SEQ ID NO:44) in all of the subunits cloned and described in Stea, A., et al. (supra) downstream from the domain I S6 transmembrane segment that is a binding site for the β subunit.
PCT publication WO 98/38301, which describes the work of the inventors herein, and which is incorporated herein by reference, reports the first description of the molecular composition of T-type calcium channel α1 subunits. The present application describes full-length genes for 3 mammalian subtypes, α1G, α1H, and α1I associated with T-type calcium channels.
In some expression systems the high threshold α1 subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits. Until recently, the reported modulatory affects of β subunit coexpression were to mainly alter kinetic and voltage-dependent properties. More recently it has been shown that β subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, et al., “Voltage-dependent facilitation of a neuronal α1C L-type calcium channel.” EMBO J (1994) 13:5032-5039; Stea, et al., “Determinants of PKC-dependent modulation of a family of neuronal calcium channels,” Neuron (1995) 15:929-940; Bourinet, et al., “Determinants of the G-protein-dependent opioid modulation of neuronal calcium channels.” Proc. Natl. Acad. Sci. (USA) (1996) 93:1486-1491.)
Because of the importance of calcium channels in cellular metabolism and human disease, it would be desirable to identify the remaining classes of α1 subunits, and to develop expression systems for these subunits which would permit the study and characterization of these calcium channels, including the study of pharmacological modulators of calcium channel function.